Proteome Fractionation

Why fractionate?

Figure 1: Schematic of plasma sample processing in typical mass spectrometric-based assay for biomarker quantitation.

The ability to accurately and sensitively quantify proteins is a fundamental requirement for proteomics to deliver biological and clinically relevant information. However, protein samples can be hugely complex and challenge analytical methods due to the vast array of unique proteins as well isoforms and post-translational modifications. Furthermore, protein concentrations may span vast range of abundances, with the range in some samples – such as human plasma – spanning 10 to 12 orders of magnitude. These challenges are further confounded as proteomics samples tend not to be analyzed as full length proteins but as enzymatically digested peptides, drastically increasing the number of entities in a mixture. A relatively simple solution to these challenges is to utilize the biochemical properties of proteins/peptides to separate complex samples into multiple, simpler fractions. There are a variety of approaches to do this, many of which may be combined to provide multiple dimensions of separation and even deeper “mining” (Figure 1).

Electrophoresis

A simple yet powerful tool is to fractionate by standard SDS-PAGE gel electrophoresis. This separates proteins by size may be easily combined with other gel electrophoresis applications such as Western blotting or immunoprecipitation to enrich or identify proteins or protein complexes of interest.

Chromatography

Strong Cation Exchange (SCX)

Figure 2: Example of SCX separation and fractionation with an increasing salt concentration showing the elution pattern of a mixture of 40 possible biomarker peptides with varying charge-based physiochemical properties.

A common method to fractionate a sample based on charge is by using Strong Cation Exchange (SCX) chromatography. In this approach peptides are loaded on to a SCX column and eluted using a high performance liquid chromatography (HPLC) salt gradient (Figure 2). This process can be automated, making it amenable for higher throughput analyses.

 

 

 

 

 

Reversed Phase

Figure 3: Example of RP pH10 separation and fractionation with an increasing organic concentration showing the elution pattern of the same mixture of 40 possible biomarker peptides with varying hydrophobicity-based physiochemical properties.

This separation differentiates peptides and proteins based upon the hydrophobic nature of each component. A gradient of increasing nonpolar solvent separates a complex mixture based upon how strongly each peptide or protein is adsorbed to the hydrophobic stationary phase. Poorly retained components contain more hydrophilic or ‘water-loving’ amino acids while those that elute later are increasingly more hydrophobic (Figure 3). Selectivity of the separation (order of elution) can be further modulated by altering the pH at which it is performed.

 

 

 

 

Offgel Isoelectric Focusing (IEF)

Isoelectric focusing (IEF) separates proteins or peptides based on their isoelectric points (pI). This is achieved by carrier ampholytes in an immobilized pH gradient gel strip but collected in 12 or 24 liquid fractions. This is a highly powerful separation technique and is easily multiplexed as several samples can be focused simultaneously.

Abundant protein depletion

Blood-derived samples such as plasma and serum are perhaps the most difficult protein samples to analyze due to the vast dynamic range of protein concentration. In a typical plasma sample, the 10 most abundant proteins will compose ~95% of total protein content. These highly abundant proteins obscure detection of the many lower abundance ones and thus sensitivity is greatly improved by depleting them. Abundant protein depletion is typically carried out by HPLC using antibody affinity columns targeting the high abundance proteins. Following depletion, samples are typically concentrated prior to downstream work.